A distinct urinary biomarker pattern characteristic of female Fabry patients that mirrors response to enzyme replacement therapy

Female patients affected by Fabry disease, an X-linked lysosomal storage disorder, exhibit a wide spectrum of symptoms, which renders diagnosis, and treatment decisions challenging. No diagnostic test, other than sequencing of the alpha-galactosidase A gene, is available and no biomarker has been proven useful to screen for the disease, predict disease course and monitor response to enzyme replacement therapy. Here, we used urine proteomic analysis based on capillary electrophoresis coupled to mass spectrometry and identified a biomarker profile in adult female Fabry patients. Urine samples were taken from 35 treatment-naïve female Fabry patients and were compared to 89 age-matched healthy controls. We found a diagnostic biomarker pattern that exhibited 88.2% sensitivity and 97.8% specificity when tested in an independent validation cohort consisting of 17 treatment-naïve Fabry patients and 45 controls. The model remained highly specific when applied to additional control patients with a variety of other renal, metabolic and cardiovascular diseases. Several of the 64 identified diagnostic biomarkers showed correlations with measures of disease severity. Notably, most biomarkers responded to enzyme replacement therapy, and 8 of 11 treated patients scored negative for Fabry disease in the diagnostic model. In conclusion, we defined a urinary biomarker model that seems to be of diagnostic use for Fabry disease in female patients and may be used to monitor response to enzyme replacement therapy.     

Effects of Bradyrhizobium japonicum and Soybean (Glycine max (L.) Merr.) Phosphorus Nutrition on Nodulation and Dinitrogen Fixation

Cells of Bradyrhizobium japonicum were grown in media containing either 1.0 mM or 0.5 μM phosphorus. In growth pouch experiments, infection of the primary root of soybean (Glycine max (L.) Merr.) by B. japonicum USDA 31, 110, and 142 was significantly delayed when P-limited cells were applied to the root. In a greenhouse experiment, B. japonicum USDA 31, 110, 122, and 142 grown with sufficient and limiting P were used to inoculate soybeans which were grown with either 5 μM or 1 mM P nutrient solution. P-limited cells of USDA 31 and 110 formed significantly fewer nodules than did P-sufficient cells, but P-limited cells of USDA 122 and 142 formed more nodules than P-sufficient cells. The increase in nodule number by P-limited cells of USDA 142 resulted in significant increases in both nodule mass and shoot total N. In plants grown with 1 mM P, inoculation with P-limited cells of USDA 110 resulted in lower total and specific nitrogenase activities than did inoculation with P-sufficient cells. Nodule numbers, shoot dry weights, and total N and P were all higher in plants grown with 1 mM P, and plants inoculated with USDA 31 grew poorly relative to plants receiving strains USDA 110, 122, and 142. Although the effects of soybean P nutrition were more obvious than those of B. japonicum P nutrition, we feel that it is important to develop an awareness of the behavior of the bacterial symbiont under conditions of nutrient limitation similar to those found in many soils.     

Trends in carbohydrate depletion, respiratory carbon loss, and assimilate export from soybean leaves at night

To evaluate assimilate export from soybean (Glycine max [L.] Merrill) leaves at night, rates of respiratory CO₂ loss, specific leaf weight loss, starch mobilization, and changes in sucrose concentration were measured during a 10-hour dark period in leaves of pod-bearing `Amsoy 71' and `Wells II' plants in a controlled environment. Lateral leaflets were removed at various times between 2200 hours (beginning dark period) and 0800 hours (ending dark period) for dry weight determination and carbohydrate analyses. Respiratory CO₂ loss was measured throughout the 10-hour dark period. Rate of export was estimated from the rate of loss in specific leaf weight and rate of CO₂ efflux. Rate of assimilate export was not constant. Rate of export was relatively low during the beginning of the dark period, peaked during the middle of the dark period, and then decreased to near zero by the end of darkness. Rate of assimilate export was associated with rate of starch mobilization and amount of starch reserves available for export. Leaves of Amsoy 71 had a higher maximum export rate in conjunction with a greater total change in starch concentration than did leaves of Wells II. Sucrose concentration rapidly declined during the first hour of darkness and then remained constant throughout the rest of the night in leaves of both cultivars. Rate of assimilate export was not associated with leaf sucrose concentration.     

Daytime and nighttime carbon balance and assimilate export in soybean leaves at different photon flux densities

To evaluate daytime and nighttime carbon balance and assimilate export in soybean (Glycine max [L.] Merrill) leaves at different photon flux densities, rates of CO₂ exchange, specific leaf weights, and concentrations of sucrose and starch were measured at intervals in leaves of pod-bearing `Amsoy 71' and `Wells II' plants grown in a controlled environment room. Assimilate export was estimated from CO₂ exchange and change in specific leaf weight. Total diurnal assimilate export was similar for both cultivars. Large cultivar differences existed, however, in the partitioning of carbon into starch reserves and the relative amounts of assimilate exported during the day and the night. Total amounts of both daytime and nighttime export increased with increasing photon flux density, as did sucrose and starch concentrations, specific leaf weight, and rate of respiratory carbohydrate loss at night. Cultivar differences in nighttime rate of export were more closely related to the differences in amount of assimilate available at the end of the day than to differences in daytime rate of net CO₂ assimilation. Daytime rates of export, however, were closely related to daytime rates of net CO₂ assimilation within each cultivar. The total amount of starch depleted during the 10-hour night increased as starch concentration at the beginning of the night increased.     

Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase

The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes.

Isocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids during postgerminative growth of oil-rich seedlings. We determined experimentally the necessary and sufficient peroxisome targeting signals (PTSs) for cottonseed, oilseed rape, and castor bean ILs in a well-characterized in vivo import system, namely, suspension-cultured tobacco (Bright Yellow) BY-2 cells. Results were obtained by comparing immunofluorescence localizations of wild-type and C-terminal-truncated proteins transiently expressed from cDNAs introduced by microprojectile bombardment. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and SRM-COOH (on oilseed rape IL) were necessary for targeting and actual import of these ILs into glyoxysomes, and ARM-COOH was sufficient for redirecting chloramphenicol acetyltransferase (CAT) from the cytosol into the glyoxysomes. Surprisingly, IL and CAT subunits without these tripeptides were still acquired by glyoxysomes, but only when wild-type IL or CAT-SKL subunits, respectively, were simultaneously expressed in the cells. These results reveal that targeting signal-depleted subunits are being piggybacked as multimers to glyoxysomes by association with subunits possessing a PTS1. Targeted multimers are then translocated through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.